Optic tissue associated with light transmission and image focusing for photoreception is formed in almost all animal life forms. The cornea is exemplary of optic tissue.
In vivo, the cornea serves to focus light and to form the anterior wall of the eye. It is composed of transparent tissue that is on average 0.55 mm thick in the central region of the cornea and 11 mm in diameter in adult humans, and is organized into six distinct regions lying parallel to the anterior and posterior surface. The structural order of these regions gives rise to the transparence of the cornea. The epithelium is located on the anterior surface of the cornea and is composed of five to six layers of cells in humans. The basal layer of the epithelium is connected via numerous attachment bodies to the basement membrane, a tightly packed filamentous layer 100 to 300 .ANG. thick. On the posterior side of this membrane, lies the stroma which accounts for nine-tenths of the thickness of the cornea in most mammals. It is composed primarily of keratocytes and, to a far lesser degree, leucocytes that lie between nearly 200 parallel layers of stroma lamellae that is formed from extracellular matrix. The outermost anterior portion of the stroma forms the Bowman's zone. Approximately 12 .mu.m thick, this zone is a cell-free layer of stroma containing extracellular matrix fibrils felted together in an irregular manner. The stroma is located between two membranes, the basement membrane as previously discussed and Descemet's membrane which is a sheet of extracellular matrix, 5 to 10 .mu.m thick, bound to the posterior side of the stroma. This second membrane is lined by the endothelium, a single layer of cells, that forms the posterior surface of the cornea. These cells are characterized by their hexagonal shape, giving the endothelium a mosaic structure. This and other information on corneal tissue is summarized by Kaufman Kaufman, H. E., McDonald, M. B., Barron, B. A., and Waltman, S. R., The Cornea (Churchill Livingstone, N.Y., 1988)!.
Within corneal tissue, the extracellular matrix serves as scaffolding to provide mechanical strength and structural organization. After being synthesized and secreted from corneal cells, the matrix forms distinct three-dimensional, lattice-like arrangements in the extracellular space of corneal tissue Komai, Y. and Ushiki, T., Invest. ophthalmol. Vis. Sci., 32, 2244 (1991)!. Considering the stroma, fibrils of extracellular matrix are interwoven to form dense felt-like sheets in Bowman's layer. At the interface between the stroma and Descemet's membrane, the matrix becomes a loose fibrillar network oriented in various directions and interlaced. In between these two regions, the stroma is composed of successively stacked layers of flat lamella bundles of matrix fibrils.
There are primarily two classes of macromolecules present in the extracellular matrix: glycosaminoglycans (GAG'S) including chondroitin sulfate and dermatan sulfate, and fibrous proteins such as fibronectin and collagen. The role of the latter group is mainly structural and adhesive. For example, fibronectin binds to cells and to other matrix macromolecules, promoting cell attachment to and subsequent migration along the matrix Alberts B., Bray, D., Lewis, J., Raff, M., Roberts, K., and Watson, J. D., Molecular Biology of the Cell (Garland Publishing, New York, 1989)!. In the cornea, GAG's regulate spacing between fibrils and the three-dimensional organization of stroma lamellae Hahn, R. A. and Birk, D. E., Development, 115, 383 (1992)!. In addition, they regulate the kinetics of fibril formation Birk, D. E. and Lande, M. A., Biochim, Biophys. Acta, 670, 362 (1981)!.
Expression and proper three-dimensional organization of extracellular matrix is essential for corneal transparency. When expression or organization is inhibited, the tissue becomes opaque. This condition occurs in patients with muscular dystrophy. The disease is characterized by improper biosynthesis of keratin sulfate, a GAG Hassell, J. R., Newsome, D. A., Krachmer, J. H., and Rodrigues, M., Proc. Natl. Acad. Sci. U.S.A., 77, 3705 (1980); Nakazawa, K., Hassell, J. R., Hascall, V. C., Lohmander, L. S., Newsome, D. A., and Krachmer, J., J. Biol. Chem., 259, 13751 (1984)!. It can also be induced chemically with agents such as .beta.-D xyoside Hahn, R. A. and Birk, D. E., Development, 115, 383 (1992)!.
Corneal transplants are the most frequently performed human transplant procedure. Since 1961, there have been more than 421,300 corneal transplantations performed in the U.S. In 1991 alone, there were 41,300 such transplantation, more than all other organ transplantations performed in that year Eye Bank Association of America, Annual Report (Washington, D.C., 1992!. Greater than 90% of corneal transplant operations successfully restore vision. The reasons for transplantation are varied. They include corneal dystrophy which results from, for example, malnutrition, dehydration and radiation exposure; keratoconus which can cause the cornea to rupture if change in corneal shape is severe; keratitis from viral or microbial sources; corneal degeneration in the elderly; chemical injury; physical trauma; transplant rejection; edema which can result from trauma to the endothelium inhibiting fluid flow between the cornea and anterior chamber; and corneal leukoma Leibowitz, H. M., Corneal Disorders: Clinical Diagnosis and Management (W. B. Saunders, Philadelphia, 1984; Brightbill, F. S., Corneal Surgery: Theory, Technique, and Tissue (Mosby-Year Book, St. Louis, 1993)!.
The majority of corneal transplantations are performed with donor tissue. The use of donor tissue results in several complications, including donor shortage. There are in excess of 5,000 patients on waiting lists for donor tissue throughout the U.S. Eye Bank Association of America, Annual Report (Washington, D.C., 1992)!. These people wait between two weeks and two years to obtain suitable tissue. And when this tissue becomes available, there is still the possibility of transplant rejection and disease transfer of HIV Salahuddin, S. Z., Palestine, A. G., Heck, E., Ablashie, D., Luckenbach, M., McCulley, J. P., and Nussenblatt, R. B., Am. J. Ophthalmol., 104, 149 (1986)!, hepatitis B Raber, I. M. and Friedman, H. M., Am. J. Ophthalmol., 104, 255 (1987)!, herpes Leibowitz, H. M., Corneal Disorders: Clinical Diagnosis and Management (W. B. Saunders, Philadelphia, 1984!, and other ailments from donor to patient. In addition to these complications, many patients with corneal disease or injury are not amenable to transplantation. This can occur, for example, when there is a chemical burn resulting in severe scarring and vascularization Brightbill, F. S., Corneal Surgery: Theory, Technique, and Tissue (Mosby-Year Book, St. Louis, 1993)!.
To overcome these difficulties, alternatives to donor tissue have been developed. One such alternative is a prosthetic implant made of an optical cylinder and supporting flange Polack, F. M. and Heimke, G., Ophthalmology, 87, 693 (1980); Trinkaus-Randall, V., Banwatt, R., Capecchi, J., Leibowitz, H. M., and Franzblau, C., Invest. Ophthalmol. Vis. Sci., 32, 3245 (1991)!. For post-operative stability, implant materials must be biocompatible and promote cell adhesion. When these two specifications are met, good vision can be retained for 7 to 8 years after implantation. Permanent stability, however, has yet to be obtained. In refractive keratoplasty procedures, a synthetic intracorneal lens can be implanted to change the refractive power of the cornea McCarey, B. E., Refract. Corneal Surg., 6, 40 (1990); Insler, M. S., Boutros, G., and Caldwell, D. R., Am. Intra-Ocular Implant Soc. J., 11, 159 (1985)!. As before, biocompatibility and cell adhesion are required for implantation to be successful, but another requirement is permeability so that nutrients can flow across the lens to the anterior portion of the cornea. Synthetic lenses have been stable in animal models for almost a decade. Clinical trials are in an early stage.
The invention described in this patent application is another alternative to the use of donor tissue for transplantation. It can be used to prepare corneal tissue from in vitro cultures of the patient's own corneal cells or from a well-defined primary culture derived from another human source. With in vitro produced tissue, shortage of tissue and disease transfer to the patient would be minimized. Also, post-operative stability should be greatly enhanced with artificially generated tissue over that currently obtained with a prosthesis or synthetic intracorneal lens.
Reported corneal tissue regeneration has been limited to date to two-dimensional models. In particular, regenerated corneal endothelial cells have been successfully transplanted into animal models Insler, M. S., and Lopez, J. G., Curr. Eye Res., 5, 967 (1986)!. Since the endothelium consists of a single layer of cells in the cornea, a two-dimensional culture can be used for transplantation. Since both the corneal epithelium and stroma are composed of multiple layers of cells, three-dimensional tissue is required to replace these cell structures when they become damaged.
To study cell structure and function in normal and abnormal cornea, three types of tissue models are currently employed: intact tissue in living animals Trinkaus-Randall, V., Banwatt, R., Capecchi, J., Leibowitz, H. M., and Franzblau, C., Invest. Ophthalmol. Vis. Sci., 32, 3245 (1991)!, donor tissue Komai, Y. and Ushiki, T. Invest. Ophthalmol. Vis. Sci., 32, 2244 (1991)!, and tissue from in vitro cell culture Geroski, D. H. and Hadley, A., Curr. Eye Res., 11, 61 (1992)!. The demand for these models is increasing yearly. The number of donor eyes that were used for research and education grew from 34,147 in 1989 to 40,239 in 1991 Eye Bank Association of America, Annual Report (Washington, D.C., 1992)!. In vitro cell culture has certain advantages over the other two models: its use avoids unnecessary loss of sight in lab animals and avoids variation in tissue characteristic of corneas from different donors. To date, the majority of cell-culture models have been two-dimensional. These models are limited in their applications, however, in that they preclude an accurate representation of three-dimensional phenomena within the cornea such as wound healing and mass transport of nutrients from the endothelium to the stroma and to the epithelium. For these phenomena, a three-dimensional model is required. Furthermore, the synergistic interaction between different cell types in a three-dimensional model could more accurately reflect the cell function in vivo than a two-dimensional model.
The use of conventional stirred or sparged bioreactors have not been generally successful for culture of three dimensional, functional tissue. Some tissues such as the Chinese hamster ovary cells grow robustly in conventional stirred bioreactors O'Connor, K. C. and Papoutsakis, E. T., Biotechnol. Tech., 6, 323 (1992)!. In contrast Sf9 fall armyworm ovary cells will not grow at all in these reactors under the same operating conditions; in fact, they die unless supplemented with a liquid surfactant Murhammer, D. W. and Goochee, C. F., Biotechnol. Prog., 6, 391 (1990)!. Only limited work has been done to date to develop three-dimensional corneal models. Bioreactors have not been used for optic tissue growth such as cornea tissue. For example, a model of multiple layers of rabbit corneal epithelial cells grown on a support of contracted collagen lattices was employed to investigate wound healing Ouyang, P. and Sugrue, S. P., "Identification of an Epithelial Protein Related to the Desmosome and Intermediate Filment Network," J. Cell Biol., Vol. 118, pages 1477-1488, 1992! rather than bioreactor produced cells.
A variety of different cells and tissues, such as bone marrow, skin, liver, pancreas, mucosal epithelium, adenocarcinoma and melanoma, have been grown in culture systems to provide three dimensional growth in the presence of a pre-established stromal support matrix. U.S. Pat. No. 4,963,489, Three-Dimensional Cell and Tissue Culture System, Naughton, et al., Oct. 16, 1990; U.S. Pat. No. 5,032,508, Three-Dimensional Cell and Tissue Culture System, Naughton, et. al., Jul. 16, 1991. A biocompatible, non-living material formed into a three dimensional structure is inoculated with stromal cells. In some cases, the three dimensional structure is a mesh pre-coated with collagen. Stromal cells and the associated connective tissue proteins naturally secreted by the stromal cells attach to and envelop the three dimensional structure. The interstitial spaces of the structure become bridged by the stromal cells, which are grown to at least subconfluence prior to inoculating the three dimensional stromal matrix with tissue-specific cells. The cells are grown on an artificial architecture rather than allowing for establishing natural organization with dimensional segregation.
The invention described herein more closely approximates intact corneal tissue than other in vitro models currently available. In the Sugrue model described above, the contracted collagen lattices serve as a synthetic extracellular matrix to which corneal cells can attached. Because these lattices do not form the intricate structure of the native matrix within corneal tissue, the Sugrue model precludes the formation of cell structures characteristic of native corneal tissue and, thus, precludes the formation of transparent tissue. In the model described in this invention, the internal structure of the tissue is more akin to native cornea. Specifically, the tissue grown on extracellular matrix synthesized by the cells themselves. There is also evidence of special organization of the matrix and cells as well as dimensional segregation in the tissue.